Attenuated Bordetella Strains

ABSTRACT

A mutated  Bordetella  strain comprising at least a mutated ptx gene, a deleted or mutated dnt gene and a heterologous ampG gene is provided. The attenuated mutated  Bordetella  strain can be used in an immunogenic composition or a vaccine for the treatment or prevention of a  Bordetella  infection. Use of the attenuated  Bordetella  strain for the manufacture of a vaccine or immunogenic composition, as well as methods for protecting mammals against infection by  Bordetella  are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S. patentapplication Ser. No. 14/860,210 filed on Sep. 21, 2015, which is acontinuation application of U.S. patent application Ser. No. 14/658,817filed on Mar. 16, 2015 (now U.S. Pat. No. 9,180,178), which is adivisional application of U.S. nonprovisional patent application Ser.No. 12/224,895 filed on Nov. 19, 2008 (now U.S. Pat. No. 9,119,804) as anational stage entry application under 35 U.S.C. 371 of internationalpatent application number PCT/EP/001942, filed on Mar. 7, 2007, whichdesignated the U.S. and claims the priority of U.S. provisional patentapplication Ser. No. 60/817,430 filed on Jun. 30, 2006 and U.S.provisional patent application Ser. No. 60/780,827 filed on Mar. 10,2006, which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to a mutated Bordetella strain comprisingat least a mutated ptx gene, a deleted or mutated-dnt gene and aheterologous ampG gene. The attenuated mutated Bordetella strain can beused in an immunogenic composition or a vaccine for the treatment orprevention of a Bordetella infection. Use of the attenuated Bordetellastrain for the manufacture of a vaccine or immunogenic compositions, aswell as methods for protecting mammals against infection by Bordetellaalso form a part of the invention.

BACKGROUND OF THE INVENTION AND RELATED PRIOR ART

Pertussis is still among the principal causes of death world-wide, andits incidence is increasing even in countries with high vaccinecoverage. Although all age groups are susceptible, it is most severe ininfants too young to be protected by currently available vaccines.

Whooping cough or pertussis is a severe childhood disease responsiblefor high mortality rates before the introduction of effective vaccinesin the second half of the 20th century. The success of these vaccineshas led to the opinion that the disease is essentially under control,although world-wide 200,000 to 400,000 pertussis-linked deaths are stillrecorded annually, and the disease still ranks sixth among the causes ofmortality due to infectious agents [1]. Although mostly prevalent indeveloping countries, the disease is also re-emerging in the developedworld [2, 3], including the U.S.A., where the incidence has increasedfive-fold over the last twenty years [4]. Unexpectedly, the epidemiologyof pertussis has changed in countries with high vaccine coverage, wherecases of adolescent and adult pertussis are increasingly frequent [5].This is probably due to progressive waning of vaccine-mediated immunityduring adolescence. Often atypical and therefore difficult to diagnose,pertussis is generally not life-threatening in adults and in many casesremains unnoticed. However, infected adults constitute an importantreservoir for transmission of the disease to very young children, tooyoung to be fully vaccinated, and therefore at risk to develop severedisease associated with high mortality rates.

Pertussis vaccination usually begins at two months of age, and fullprotection requires at least three immunizations at one- to two-monthintervals. Therefore, infants are not fully protected before the age of6 months using the currently available vaccines. To reduce the incidenceof pertussis in the very young and most vulnerable age groups, earlyimmunization, possibly at birth, would thus be highly desirable.However, numerous studies in humans and in animal models have suggestedthat the neonatal immune system is too immature to effectively inducevaccine-mediated protective immunity [6, 7]. Especially the IFN-γproduction, indicative of a Th1 response that is essential to thedevelopment of protective immunity to pertussis [8], appears to besignificantly reduced in human newborns, compared to older children oradults [9]. This is also reflected by the fact that significant amountsof antigen-specific IFN-γ are only produced after several months (≧6months) in children vaccinated with pertussis vaccines, especially withacellular vaccines (aPV) [10].

Natural infection with Bordetella pertussis has long been considered toinduce strong and long-lasting immunity, that wanes much later thanvaccine-induced immunity [5, 11]. Furthermore, infection with B.pertussis induces measurable antigen-specific Th1 type immune responseseven in very young children (as young as one month of age) [12]. Theseobservations suggest that live vaccines applicable by the nasal route inorder to mimic as closely as possible natural infection, may beattractive alternatives over the currently available vaccines.

There are many vaccinating compositions to treat Bordetella infectionsknown in the art. However, these immunogenic compositions are not usedto treat newborn children or in cases where an epidemic and rapidprotective immunity is required.

Thus, French Patent FR 0206666 discloses live Bordetella strains thathave been rendered deficient in at least two toxins chosen from PTX,DNT, AC and TCT. This patent discloses the over expression of anendogenous ampG gene by the addition of a strong promoter, and theaddition of 11 terminal amino acids of the ampG gene from E. coli.

Mielcarek et al, Vaccine (2006; 2452: 52154-52-55) disclose a strain ofBordetella pertussis attenuated of PTK, DTN- and TCr for use in theimmunization of mice. This reference discloses that to reduce theproduction of tracheal cytotoxin, the ampG gene should be overexpressed.However, upon further evaluation, the authors realized that byover-expressing the ampG gene, there is an increase in trachealcytotoxin and not a decrease as was originally thought.

Mielcarek et al in Advance Drug Delivery Review 51 (2001) pgs. 55-69disclose that live vaccines can induce systemic and mucosal responseswhen administered by the oral or nasal route.

Roduit et al in Infection and Immunity (2002 July; 70(7): 3521-8)describe vaccinating neonatals and infants with mutated Bordetellastrains with a DTP composition.

Mattoo et al, in Frontiers of Bioscience 6, e168-e186 (2001), suggestreplacing the endogenous ampG gene in Bordetella with the E. coli ampGgene, which resulted in a decrease in the amount of TCT produced.

Thus, the prior art although disclosing various types of vaccinatingcompositions fails to address the problem of providing a vaccine orimmunogenic composition that can provide protection to a newborn priorto six months. Furthermore, the prior art fails to disclose animmunogenic or a vaccine that provides rapid protective immunity againsta Bordetella infection. The prior art also fails to disclose animmunogenic composition or vaccine that provides a rapid protectiveimmunity against a Bordetella infection, said protective immunityincreasing over at least the next two months following vaccination.

Therefore, it is an object of the present invention to overcome thedeficiencies in the prior art.

It is another object of the present invention to produce a liveattenuated vaccine candidate or immunogenic composition through geneticattenuation of a Bordetella strain such as B. pertussis or B.parapertussis to diminish pathogenicity, while maintaining the abilityto colonize and induce protective immunity.

It is another object of the present invention to produce a vaccine orimmunogenic composition that induces protection in newborns after asingle intranasal administration that is superior to the protectionprovided by the current aPV.

It is yet another object of the present invention to provide protectionagainst infection with Bordetella parapertussis, as well as Bordetellapertussis which was not seen after vaccination with aPV.

Another object of the present invention is to induce strong protectiveimmunity in newborns against Bordetella infection.

Yet another object of the present invention is to provide a vaccine orimmunogenic composition that induces mucosal and systemic immunity.

It is another object of the present invention to produce a liveattenuated Bordetella pertussis strain to be given as a single-dosenasal vaccine in early life, called BPZE1.

It is yet another object of the present invention to provide a vaccinethat can not only be used to vaccinate newborns, but can be used in allmammals of any age in the case of an epidemic of whooping cough.

Another object of the present invention is to provide a vaccine againstBordetella infection that induces a rapid protective immunity and/or aprotective immunity that increases over at least the next two monthsafter the vaccination.

Yet another object of the present invention is to provide prevention ortreatment against Bordetella infection that is relatively low inproduction costs.

These and other objects are achieved by the present invention asevidenced by the summary of the invention, description of the preferredembodiments and the claims.

SUMMARY OF THE INVENTION

The present invention provides a mutated Bordetella strain comprising atleast a mutated pertussis toxin (ptx) gene, a deleted or mutateddermonecrotic toxin (dnt) gene, and a heterologous ampG gene.

In another aspect the present invention relates to an immunogeniccomposition comprising a mutated Bordetella strain comprising at least amutated pertussis toxin (ptx) gene, a deleted or mutated pertussisdermonecrotic toxin (dnt) gene, and a heterologous ampG gene.

In yet another aspect the present invention provides a vaccinecomprising the attenuated Bordetella strain comprising at least amutated pertussis toxin (ptx) gene, a deleted or mutated pertussisdermonecrotic toxin (dnt) gene, and a heterologous ampG gene.

In still another aspect, the present invention provides the use of anattenuated Bordetella strain comprising at least a mutated ptx gene, adeleted or mutated dnt gene, and a heterologous ampG gene for themanufacture of a vaccine for the prevention of a Bordetella infection.

In yet another aspect, the present invention provides the use of anattenuated Bordetella strain comprising at least a mutated ptx gene, adeleted or mutated dnt gene, and a heterologous ampG gene for themanufacture of a vaccine for the induction of an immune responsedirected preferentially toward the Th1 pathway against said attenuatedBordetella.

Also provided is a method of protecting a mammal against disease causedby infection by Bordetella pertussis and Bordetella parapertussiscomprising administering to said mammal in need of such treatment amutated Bordetella strain comprising at least a mutated ptx gene, adeleted or mutated dnt gene, and a heterologous ampG gene.

A method of providing a rapid protective immunity against a Bordetellainfection comprising administering to said mammal in need of suchtreatment a mutated Bordetella strain comprising at least a mutated ptxgene, a deleted or mutated dnt gene, and a heterologous ampG gene isalso part of the present invention.

A method of providing a rapid protective immunity against a Bordetellainfection comprising administering to a mammal in need of such treatmenta mutated Bordetella strain comprising at least a mutated ptx gene, adeleted or mutated dnt gene, and a heterologous ampG gene or a vaccinecomprising said mutated Bordetella strain, wherein said method providesfurther an increase in said protective immunity over at least two monthsafter vaccination is still another aspect of the present invention.

Use of the mutated Bordetella strain comprising at least a mutated ptxgene, a deleted or mutated dnt gene and a heterologous ampG gene for thepreparation of a multivalent vaccine (i.e., a vaccine for preventing ortreating infections caused by different pathogens) to treat respiratorydiseases is yet another aspect of the present invention.

Use of an attenuated Bordetella strain of the invention, byadministration to mammals in need of a rapid protective immunity againsta Bordetella infection, wherein said protective immunity increases overat least two months after administration, is also part of the presentinvention.

A method to provide a mucosal response and a systemic response to treator protect against Bordetella infections in mammals is still anotheraspect of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph illustrating the TCT present in culturesupernatants of BPSM and BPZE expressed as means of nM/OD_(540nm)(±standard error) of 3 separate cultures for each strain.

FIG. 2 is an immunoblot analysis of PTX production in the culturesupernatants of BPSM (lane 1) and BPZE (lane 2). The sizes of the Mrmarkers are expressed in kDa and given in the left margin.

FIG. 3 is a Southern-blot analysis of the dnt locus in BPSM (lane 1) andBPZE (lane 2). The lengths of the size markers are indicated in basepairs (bp) are shown in the left margin.

FIG. 4 is a graph illustrating the growth rates of BPSM (black line) andBPZE1 (dotted line) in liquid culture.

FIG. 5 are electron micrographs representative of BPSM (left) and BPZE1(right) grown in liquid medium for 24 h.

FIG. 6 is a graph illustrating the in vitro adherence of BPSM (blackcolumns) and BPZE1 (white columns) to human pulmonary epithelial A549cells (left) and murine macrophage-like J774 cells (right). The resultsare expressed as means of percentages of binding bacterial relative tothe bacteria present in the inoculum from three different experiments.

FIG. 7 is a graph illustrating lung colonization by BPSM (black lines)and BPZE1 (dotted lines) of adult mice infected intranasally with 106CFU of BPZE1 or BPSM. The results are expressed as mean (±standarderror) CFUs from three to four mice per group and are representative oftwo separate experiments. P=0.004.

FIG. 8 are photographs of a histological analysis of lungs from BPZE1(upper panel) or BPSM-infected (middle panel) adult mice compared tocontrols given PBS (lower panel). One week after infection, the lungswere aseptically removed and fixed in formaldehyde. Sections werestained with hematoxylin and eosin and examined by light microscopy.

FIG. 9 are graphs illustrating the protection against B. pertussis in(a) adult and (b) infant mice or B. parapertussis in infant mice (d).Mice immunized with BPZE1, aPV or PBS (naive) were challenged with BPSM(a and b) or B. parapertussis (d), and lung CFU counts were determined 3h (white bars) or 7 days (black bars) later. Results are expressed asmean (±standard error) CFUs from 3-4 mice per group and arerepresentative of two separate experiments. (b,*, P=0.009; d,*, P=0.007)(c) CFU counts 3 h after BPSM challenge in adult mice vaccinated withBPZE1 or aPV, compared to controls. Results obtained from 3 separateexperiments are expressed as percentages of CFUs of each mouse relativeof the average of CFUs in non-immunized group from the same experiment.

FIG. 10 are bar graphs illustrating the immune responses induced byBPZE1 or aPV immunization. (a) Anti-FHA lgG(H+1) titers and (b)lgG1/IgG2a ratios before (white bars) or 1 week after BPSM challenge(black bars) in BPZE1 or aPV immunized mice, compared to controls. (c)IFN-y to IL-5 ratios produced by FHA-, PTX- or ConA-stimulatedsplenocytes from mice vaccinated 2 months before with BPZE1 (black bars)or aPV (white bars), compared to controls (gray bars). Antibodies andcytokines were measured in individual mice, and the results areexpressed as mean values (±standard error) for 4 mice per group testedin triplicate.

FIG. 11 is the amino acid sequence of pertussis toxin (SEQ 10 NO:1)(islet-activating protein S1). The first 34 amino acids are the signalsequence, while amino acids 35 to 269 are the mature chain.

FIG. 12 is the amino acid sequence of dennonecrotic toxin (SEQ ID NO:2).

FIG. 13 is the amino acid sequence of .AmpG from Bordetella pertussis(SEQ ID NO:3).

FIG. 14 is the amino acid sequence of AmpG from Escherichia coli (SEQ IDNO:4).

DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE PRESENT INVENTION

As used herein, the abbreviation “PTX” refers to pertussis toxin, whichsynthesizes and secretes an ADP-ribosylating toxin. PTX is composed ofsix polypeptides S1 to S5, the enzymatically active moiety is called S1.PTX has a 34 amino acid signal sequence, while the mature chain consistsof amino acids 35 to 269. PTX is the major virulence factor expressed byB. pertussis. The A moiety of these toxins exhibitADP-ribosyltransferase activity and the B portion mediates binding ofthe toxin to host cell receptors and the translocation of A to its siteof action (57).

As used herein the abbreviation “DNT” refers to pertussis dermonecrotictoxin, which is a heat labile toxin that induces localized lesions inmice and other laboratory animals when it is injected intradermally. Itis lethal to mice when it is injected in low doses intravenously (58 to61). DNT is considered to be a virulence factor for the production ofturbinate atrophy in porcine atrophic rhinitis (62, 63).

As used herein the abbreviation “TCT” refers to tracheal cytotoxin,which is a virulence factor synthesized by Bordetellae. TCT is apeptidoglycan fragment and has the ability to induce interleukin-1production and nitric oxide synthase. It has the ability to cause stasisof cilia and has lethal effects on respiratory epithelial cells.

The term “mammal” encompasses any of various warm-blooded vertebrateanimals of the class Mammalia, including humans, characterized by acovering of hair on the skin and, in the female, milk-producing mammaryglands for nourishing the young.

The term “attenuated” means a weakened, less virulent Bordetella strainthat is capable of stimulating an immune response and creatingprotective immunity, but does not cause any illness.

The terminology “rapid protective immunity” means that immunity againstBordetella is conferred in a short time after administration of themutated Bordetella strain of the present invention. By “short time”means vaccinated and challenged one week later. More specifically, thereis a quick expansion of existing pathogen-specific peripherallymphocytes, CDS+ cytotoxic effectors (CTLs) and CD4+ helper cells. TheCD4+ helper cells induce B cell maturation and antibody production.Thus, lymphocytes with the memory pool are poised to rapidly proliferateat the time of subsequent infection.

The term “Bordetella strain” encompasses strains from Bordetellapertussis, Bordetella parapertussis and Bordetella bronchiseptica.

The expression “Bordetella infection” means an infection caused by atleast one of the three following strains: Bordetella pertussis,Bordetella parapertussis and Bordetella bronchiseptica.

By “child” is meant a person or a mammal between 6 months and 12 yearsof age.

By the term “newborn” is meant a person or a mammal that is between 1day old and 24 weeks of age.

The term “treatment’ as used herein is not restricted to curing adisease and removing its causes but particularly covered means to cure,alleviate. Remove or lessen the symptoms associated with the disease ofinterest, or prevent or reduce the possibility of contracting anydisorder or malfunction of the host body.

The terms “protection” and “prevention” are used herein interchangeablyand mean that an infection by Bordetella is impeded.

“Prophylaxis vaccine” means that this vaccine prevents Bordetellainfection upon future exposure.

By “preferentially towards the Th1 pathway” is meant that the Th1pathway is favored over the Th2 pathway.

The term “immunogenic composition” means that the composition can inducean immune response and is therefore antigenic. By “immune response”means any reaction by the immune system. These reactions include thealteration in the activity of an organism immune system in response toan antigen and may involve, for example, antibody production, inductionof cell-mediated immunity, complement activation or development ofimmunological tolerance

More specifically, the present invention provides at least a triplemutated Bordetella strain that can be used as an immunogenic compositionor a vaccine. It will be appreciated that the at least triple mutatedBordetella strain contains a mutated ptx gene, a deleted or mutated dntgene and a heterologous ampG gene. The heterologous ampG gene productreduces in large quantities the amount of tracheal cytotoxin that isproduced.

The present invention is not limited to only the triple mutantsdescribed above. Other additional mutations can be undertaken such asadenylate cyclase (AC) deficient mutants (64), lipopolysaccharide (LPS)deficient mutants (65), filamentous hemagglutinin (FHA) (66) and any ofthe bvg-regulated components (67).

The starting strain which is mutated can be any Bordetella strainincluding Bordetella pertussis, Bordetella parapertussis and Bordetellabronchiseptica. In one aspect the starting strain used to obtain themutated Bordetella strain is B. pertussis.

The construction of the mutated Bordetella strain starts with replacingthe Bordetella ampG gene in the strain with a heterologous ampG gene.Any heterologous ampG gene can be used in the present invention. Theseinclude all those gram-negative bacteria that release very small amountsof peptidoglycan fragments into the medium per generation. Examples ofgram-negative bacteria include, but are not limited to Escherichia coli,Salmonella, Enterobacteriaceae, Pseudomonas, Moraxella, Helicobacter,Stenotrophomonas, Legionella and the like.

By replacing the Bordetella ampG gene with a heterologous ampG gene, theamount of tracheal cytoxin (TCT) produced in the resulting strainexpresses less than 1% residual TCT activity. In another embodiment, theamount of TCT toxin expressed by the resulting strain is between 0.6% to1% residual TCT activity or 0.4% to 3% residual TCT activity or 0.3% to5% residual TCT activity.

PTX is a major virulence factor responsible for the systemic effects ofB. pertussis infections, as well as one of the major protectiveantigens. Due to its properties, the natural ptx gene is replaced by amutated version so that the enzymatically active moiety S1 codes for anenzymatically inactive toxin, but the immunogenic properties of thepertussis toxin are not affected. This can be accomplished by replacingthe arginine (Arg) at position 9 of the sequence with a lysine (Lys).Furthermore, a glutamic acid (Glu) at position 129 is replaced with aglycine (Gly).

Other mutations can also be made such as those described in U.S. Pat.No. 6,713,072, incorporated herein by reference, as well as any known orother mutations able to reduce the toxin activity to undetectablelevels. Allelic exchange is first used to delete the ptx operon and thento insert the mutated version.

Finally, the dnt gene is then removed from the Bordetella strain byusing allelic exchange. Besides the total removal, the enzymaticactivity can also be inhibited by a point mutation. Since DNT isconstituted by a receptor-binding domain in the N-terminal region and acatalytic domain in the C-terminal part, a point mutation in the dntgene to replace Cys-1305 to Ala-1305 inhibits the enzyme activity of DNT(68). DNT has been identified as an important toxin in Bordetellabronchiseptica and displays lethal activity upon injection of minutequantities (26).

Besides allelic exchange to insert the mutated ptx gene and theinhibited or deleted dnt gene, the open reading frame of a gene can beinterrupted by insertion of a genetic sequence or plasmid. This methodis also contemplated in the present invention.

The triple mutated strain of the present invention is called a BPZE1strain and has been deposited with the Collection Nationale de Culturesde Micrororganismes (CNCM) in Paris, France on Mar. 9, 2006 under thenumber CNCM 1-3585. The mutations introduced into BPZE1 result indrastic attenuation, but allow the bacteria to colonize and persist.Thus, in another embodiment the present invention provides BPZE1, whichcan induce mucosal immunity and systemic immunity when administered. Inanother aspect the BPZE1 is administered intranasally.

The mutated Bordetella strains of the present invention can be used inimmunogenic compositions. Such immunogenic compositions are useful toraise an immune response, either an antibody response and or preferablya T cell response in mammals. Advantageously, the T cell response issuch that it protects a mammal against Bordetella infection or againstits consequences.

The mutated Bordetella strains of the present invention can be used aslive strains or chemically or heat-killed strains in the vaccines orimmunogenic compositions. In one aspect, the live strains are used fornasal administration, while the chemically—or heat killed strains can beused for systemic or mucosal administration.

The immunogenic composition may further comprise a pharmaceuticallysuitable excipient or carrier and/or vehicle, when used for systemic orlocal administration. The pharmaceutically acceptable vehicles include,but are not limited to, phosphate buffered saline solutions, distilledwater, emulsions such as an oil/water emulsions, various types ofwetting agents sterile solutions and the like.

The immunogenic composition of the invention can also compriseadjuvants, i.e., any substance or compound capable of promoting orincreasing a T-cell mediated response, and particularly a CD4⁺-mediatedor CD8⁺-mediated immune response against the active principle of theinvention. Adjuvants such as muramyl peptides such as MDP. IL-12,aluminium phosphate, aluminium hydroxide, Alum and/or Montanide® can beused in the immunogenic compositions of the present invention.

It would be appreciated by the one skilled in the art that adjuvants andemulsions in the immunogenic compositions are used when chemically orheat treated mutated Bordetella strains are used in the vaccines orimmunogenic compositions.

The immunogenic compositions of the invention further comprise at leastone molecule having a prophylactic effect against a Bordetella infectionor the detrimental effects of Bordetella infection, such as a nucleicacid, a protein, a polypeptide, a vector or a drug.

The immunogenic composition of the invention is used to elicit a T-cellimmune response in a host in which the composition is administered. Allimmunogenic compositions described above can be injected in a host viadifferent routes: subcutaneous (s.c.), intradermal (i.d.), intramuscular(i.m.) or intravenous (i.v.) injection, oral administration andintranasal administration or inhalation.

When formulated for subcutaneous injection, the immunogenic compositionor vaccine of the invention preferably comprises between 10 and 100 μgof the Bordetella strain per injection dose, more preferably from 20 to60 μg/dose, especially around 50 μg/dose, in a sole injection.

When formulated for intranasal administration, the Bordetella strain isadministered at a dose of approximately 1×10³ to 1×10⁶ bacteria,depending on the weight and age of the mammal receiving it. In anotheraspect a dose of 1×10⁴ to 5×10⁶ can be used.

The mutated Bordetella strains of the present invention can be used asan attenuated vaccine to protect against future Bordetella infection. Inthis regard, an advantage of the present invention is that a single dosecan be administered to mammals and the protection can last at least fora duration of longer than two months, particularly longer than sixmonths. The vaccine of the present invention can be administered tonewborns and protects against infection of whooping cough. This isespecially crucial since the fatality rate from Bordetella pertussisinfections is about 1.3% for infants younger than 1 month.

Moreover, the vaccines of the present invention can be used in adultmammals when there is an epidemic or in older adults over the age of 60,since their risk of complications maybe higher than that of olderchildren or healthy adults.

The vaccines can be formulated with the physiological excipients setforth above in the same manner as in the immunogenic compositions. Forinstance, the pharmaceutically acceptable vehicles include, but are notlimited to, phosphate buffered saline solutions, distilled water,emulsions such as an oil/water emulsions, various types of wettingagents sterile solutions and the like. Adjuvants such as muramylpeptides such as MDP. IL-12, aluminium phosphate, aluminium hydroxide,Alum and/or Montanide® can be used in the vaccines.

The vaccines of the present invention are able to induce high titers ofserum IgG against FHA. The analysis of the antigen-specific cytokinepatterns revealed that administration with the mutated attenuatedBordetella strains of the present invention favored a strong TH1response.

The vaccines of the present invention provide high level of protectionagainst a Bordetella infection i.e., a level of protection higher than90%, particularly higher than 95%, more particularly higher than 99%(calculated 7 days after infection as detailed on example 9). The levelof protection of the vaccine comprising the BPZE1 strain reaches morethan 99.999% compared to non-vaccinated (naïve) mice, at least twomonths after vaccination.

The vaccines can be administered subcutaneous (s.c.), intradermal(i.d.), intramuscular (i.m.) or intravenous (i.v.) injection, oraladministration and intranasal administration or inhalation. Theadministration of the vaccine is usually in a single dose.Alternatively, the administration of the vaccine of the invention ismade a first time (initial vaccination), followed by at least one recall(subsequent administration), with the same strain, composition orvaccine, or with acellular vaccines, or a combination of both.

In one aspect, intranasal administration or inhalation of the vaccinesis accomplished, which type of administration is low in costs andenables the colonization by the attenuated strains of the invention ofthe respiratory tract: the upper respiratory tract (nose and nasalpassages, paranasal sinuses, and throat or pharynx) and/or therespiratory airways (voice box or larynx, trachea, bronchi, andbronchioles) and/or the lungs (respiratory bronchioles, alveolar ducts,alveolar sacs, and alveoli)

Intranasal administration is accomplished with an immunogeniccomposition or a vaccine under the form of liquid solution, suspension,emulsion, liposome, a cream, a gel or similar such multiphasiccomposition. Solutions and suspensions are administered as drops.Solutions can also be administered as a fine mist from a nasal spraybottle or from a nasal inhaler. Gels are dispensed in small syringescontaining the required dosage for one application.

Inhalation is accomplished with an immunogenic composition or a vaccineunder the form of solutions, suspensions, and powders; theseformulations are administered via an aerosol or a dry powder inhaler.Compounded powders are administered with insufflators or puffers.

Use of the mutated Bordetella strains comprising at least a mutated ptxgene, a deleted or mutated dnt gene and a heterologous ampG gene for thepreparation of a multivalent vaccine to treat respiratory diseases isyet another aspect of the present invention. In this regard, theattenuated mutated Bordetella strain described above, can be used as aheterologous expression platform to carry heterologous antigens to therespiratory mucosa. Thus, such respiratory pathogens such as Neisseria,Pneumophila, yersinia, pseudomonas, mycobacteria, influenza and the likecan prevent infection using the BPZE1 as a carrier.

Use of the live attenuated mutated Bordetella strains described hereinfor the manufacture of a vaccine for the treatment or prevention ofBordetella infection is also encompassed by the present invention. Inthis regard, the vaccine can be used for the simultaneous treatment orprevention of an infection by B. pertussis and B. parapertussis.

Use of the vaccine to provide rapid protective immunity in case of apertussis epidemic is also encompassed by the present invention.

Use of the vaccine to provide a rapid protective immunity, increasingover the at least next two months following vaccination is alsoencompassed by the present invention.

The vaccine or immunogenic composition is also provided in a kit. Thekit comprises the vaccine or immunogenic composition and an informationleaflet providing instructions for immunization.

The present invention also relates to a method for inducing T-cellmediated immune response and particularly a CD4⁺-mediated immuneresponse or a CD8⁺-mediated immune response, comprising administeringthe live attenuated Bordetella strains of the invention in a non-humanmammal or a human mammal.

A method of protecting a mammal against disease caused by infection byBordetella comprising administering to said mammal in need of suchtreatment a mutated Bordetella strain comprising at least a mutated ptxgene, a deleted or mutated dnt gene, and a heterologous ampG gene isanother embodiment of the present invention. This method encompassestreating or preventing infections against Bordetella pertussis and/orBordetella parapertussis. In one aspect the BPZE1 strain is used in thismethod.

Also a method of providing a rapid protective immunity against aBordetella infection comprising administering to said mammal in need ofsuch treatment a mutated Bordetella strain comprising at least a mutatedptx gene, a deleted or mutated dnt gene, and a heterologous ampG gene isencompassed by the present invention. In one aspect the BPZE1 strain isused in this method.

Moreover, the mutated live attenuated Bordetella strains of the presentinvention induce mucosal immunity, as well as systemic immunity. Thus,in another aspect the invention also relates to a method of inducingmucosal and systemic immunity by administering to a mammal in need ofsuch treatment the mutated live attenuated Bordetella strains of thepresent invention. In one aspect the BPZE1 strain is used in thismethod.

Besides its role in the prevention and/or treatment of Bordetellainfection, the mutated strain of the invention may be used as vector, tobear at least one further heterologous nucleic acid sequence encoding aRNA (such as antisense RNA) or a protein of interest. This means thatthe mutated strain bears at least one further heterologous nucleic acidsequence in addition to the heterologous ampG gene. In one aspect, theprotein encoded by this at least one further heterologous nucleic acidsequence is a protein for which the expression is desired in therespiratory tract. In another aspect, the protein of interest is anantigen, such as a viral, a bacterial or a tumoral antigen, againstwhich an immune response is desired. Therefore, the mutated Bordetellastrain bearing at least one further heterologous nucleic acid sequencemay also be used as a vaccine. The definitions given above foradministration of the vaccine or immunogenic composition also apply to avaccine comprising mutated Bordetella strain bearing at least onefurther heterologous nucleic acid sequence. Examples of heterologousproteins are antigens of pathogens causing infections of or diseasesassociated with the respiratory track: poliomyelitis, influenza(influenzavirus from Orthomyxoviridae family) or antigens frompneumococcus (such as Streptococcus pneumoniae).

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.

EXAMPLES Materials and Methods Example 1—Bordetella Strains and GrowthConditions

The B. pertussis strains used in this study were all derived from B.pertussis BPSM 1131, and B. parapertussis is a streptomycin-resistantderivative of strain 12822 (kindly provided by Dr. N. Guiso, LnstitutPasteur Paris, France). All Bordetella strains were grown onBordet-Gengou (BG) agar (Difco, Detroit, Mich.) supplemented with 1%glycerol, 20% defibrinated sheep blood, and 100 Lg/ml streptomycin. Forcell adherence assays, exponentially growing B. pertussis was inoculatedat an optical density of 0.15 at 600 nm in 2.5 ml modifiedStainer-Scholte medium [14] containing 1 g/l heptakis(2,6-di-o-methyl)β-cyclodextrin (Sigma) and supplemented with 65 μCi/ml L-[³⁵S]methionineplus L-[³⁵S]cysteine (NEN, Boston, Mass.) and grown for 24 h at 37° C.The bacteria were then harvested by centrifugation, washed three timesin phosphate-buffered saline (PBS) and resuspended in RPMI 1640 (Gibco,Grand Island, N.Y.) at the desired density.

Example 2—Construction of B. pertussis BPZE1

To construct B. pertussis BPZE1, the B. pertussis ampG gene was replacedby Escherichia coli ampG using allelic exchange. A PCR fragment namedmet and located at position 49,149 to 49.990 of the B. pertussis genome(http://www.sanger.ac.uk/Projects/B_pertussis/), upstream of the B.pertussis ampG gene, was amplified using oligonucleotides A:5′-TATAAATCGATATTCCTGCTGGTTTCGTTCTC-3′ (SEQ ID No:5) and B:5′-TATAGCTAGCAAGTTGGGAAACGACACCAC-3′ (SEQ ID No:6), and B. pertussisBPSM [13] genomic DNA as a template. This 634 bp fragment was insertedinto Topo PCRII (InVitrogen Life Technology, Groningen. The Netherlands)and then excised as a ClaI-NheI fragment and inserted into ClaI- andNheI-digested pBP23 [50], a suicide vector containing the E. coli ampGgene with flanking B. pertussis DNA of 618 bp (from position 50,474 to51,092 of the B. pertussis genome) and 379 bp (from position 52,581 to52.960 of the B. pertussis genome) at the 5′ and 3′ end of E. coli ampG,respectively. The resulting plasmid was transferred into E. coli SM10[51], which was then conjugated with BPSM, and two successive homologousrecombination events were selected as described [52]. Ten individualcolonies were screened by PCR as follows. The colonies were suspended in100 μl H₂O, heated for 20 min. at 95° C., and centrifuged for 5 min at15,000×g. One μl of supernatants was then used as template for PCR usingoligonucleotides A and C: 5′-TAAGAAGCAAAATAAGCCAGGCAIT-3′ (SEQ ID No:7)to verify the presence of E. coli ampG and using oligonucleotides D:5′-TATACCATGGCGCCGCTGCTGGTGCTGGGC-3′ (SEQ ID No:8) and E:5′-TATATCTAGACGCTGGCCGTAACCTTAGCA-3′ (SEQ ID No:9) to verify the absenceof B. pertussis ampG. One of the strains containing E. coli ampG andlacking B. pertussis ampG was then selected, and the entire ampG locuswas sequenced. This strain was then used for further engineering.

The ptx genes were deleted from the chromosome of this strain asdescribed [21] and then replaced by mutated ptx coding inactive PTX. TheEcoRI fragment containing the mutated ptx locus from pPT-RE [16] wasinserted into the EcoRI site of pJQ200mp18rpsl [53]. The resultingplasmid was integrated into the B. pertussis chromosome at the ptx locusby homologous recombination after conjugation via E. coli SM10. The ptxlocus in the chromosome of the resulting B. pertussis strain wassequenced to confirm the presence of the desired mutations. Toxinproduction was analyzed by immunoblotting using a mix of monoclonalantibodies IB7 [54] specific for subunit S1, and 11E6 [55] specific forsubunits S2 and S3 of PTX.

Finally, the dnt gene was deleted from the resulting B. pertussis strainas the dnt flanking regions were amplified by PCR using BPSM genomic DNAas a template and oligonucleotides F:5′-TATAGAATTCGCTCGGTCGCTGGTCAAGG-3′ (SEQ ID No:10) and G:5′-TATATCTAGAGCAATGCCGATTCATCTTTA-3′ (SEQ ID No:11) for the dnt upstreamregion, and H: 5′-TATATCTAGAGCGGCCTT TATTGCTITTCC-3′ (SEQ ID No:12) andI: 5′-TATAAAGCTTCTCATGCACGCCG GCTTCTC-3′ (SEQ ID No:13) for the dntdownstream region, as primers. The resulting 799-bp and 712-bp DNAfragments were digested with EcoRI/XbaI and XbaI/HindIII, respectively,and linked together using the Fast Link kit (Epicentre Biotechnologies,Madison, Wis.). The ligated fragment was amplified by PCR usingoligonucleotides F and 1, and the 1505-bp PCR fragment was then insertedinto pCR2.1-Topo (Invitrogen), re-isolated from the resulting plasmid asan EcoRI fragment and inserted into the unique EcoRI site ofpJQmp200rpsL18. The resulting plasmid was introduced into B. pertussisby conjugation via E. coli SM10, Successful deletion of the dnt gene byallelic exchange was verified by Southern blot analysis onPvuII-digested B. pertussis genomic DNA using the PCR fragmentcorresponding to the dnt upstream region as a probe. The probe waslabeled with digoxigenin (DIG) using the DIG Easy Hyb labeling kit(Roche, Meylan, France). The sizes of the hybridizing bands weredetermined from the migration distance of the Dig-labeled DNA molecularmarker III (Roche). The dnt locus of this final strain, named BPZE1 wassequenced.

Example 3—Analysis of TCT Production

For sensitive quantitation of TCT production, culture supernatants of B.pertussis grown to logarithmic phase were collected, subjected to solidphase extraction [15] and derivatized with phenylisothiocyanate (PITC,Pierce). The resulting phenylthiocarbamyl (PTC) derivatives wereseparated by reversed-phase HPLC using a C8 column (Perkin Elmer) anddetected at 254 nm. The amount of B. pertussis PTC-TCT in each samplewas determined by comparing the peak area and elution time with anidentically processed TCT standard.

Example 4—Cell-Adherence Assay

To analyze adherence properties of the B. pertussis strains, theirattachment rates to the human pulmonary epithelial cell line A549 (ATCCn^(o) CCL-185) and the murine macrophage cell line J774 (ATCC n^(o)TIB-67) were measured as previously described [16].

Example 5—Transmission Electron Microscopy

The single droplet-negative staining procedure was used as describedpreviously [17] with the following modifications. 20 μl of a suspensionat approximately 10⁹ bacteria/ml were absorbed for 2 min. onto formformvard carbon-coated nickel grids (400 mesh; Electron MicroscopySciences EMS, Washington, Pa.). After 30 seconds air-drying the gridswere stained for 2 minutes with 20 μl of 2% phosphotungstic acid (pH7;EMS) and examined after air-drying under a transmission electronmicroscope (Hitachi 7500, Japan) at 60 kvolts and high resolution.

Example 6—Intranasal Infection and Vaccination

3-week and 8-week old female Balb/C were kept under specificpathogen-free conditions, and all experiments were carried out under theguidelines of the Institut Pasteur de Lille animal study board. Micewere intranasally infected with approximately 4×10⁶ bacteria in 20 μlPBS, and kinetics of CFU in the lungs were measured as previouslydescribed [18]. For vaccination with aPV (Tetravac; Aventis-Pasteur,France), mice were immunized intraperitoneally (i.p.) with 20% of thehuman dose and boosted one month later using the same dose.

Example 7—Antibody Determination

Sera were collected, and antibody titers were estimated by enzyme-linkedimmunosorbent assays (ELISA) as previously described [18].

Example 8—Cytokine Assays

Spleen cells from individual mice were tested at different time pointsafter immunization for in vitro cytokine production in response toheat-killed B. pertussis BPSM (10⁶ cells/ml), 5.0 μg/ml PTX (purifiedfrom B. pertussis BPGR4 [19] as previously described [20] andheat-inactivated at 80° C. for 20 min). 5.0 μg filamentous hemagglutinin(FHA, purified from B. pertussis BPRA [12] as previously described[22]), 5 μg/ml concanavalin A (Sigma Chemical Co., St. Louis, Mo.) ormedium alone as control. Supernatants were removed from triplicatecultures after 72 h incubation at 37° C. and 5% CO₂, and IFN-γ and IL-5concentrations were determined by immunoassays (BD OptEIA set,Pharmingen).

Example 9—Intranasal Infection and Vaccination: Challenge at 1, 2, 3 and4 Weeks

An infant (3 weeks-old) mouse model 1291 was used to compare theefficiency of vaccination with BPZE1 with the one of vaccination withacellular pertussis vaccine (aPv). Female Balb/C mice were intranasallyinfected with approximately 1×10⁶ BPZE1 strain in 20 μl PBS. Forvaccination with aPv (Tetravac; Aventis-Pasteur, France), mice wereimmunized intraperitoneally with 20% of the human dose. One, two, threeor four weeks after vaccination with BPZE1 or aPv, mice wereintranasally challenged with virulent B. pertussis BPSM/bctA-lacZ strain[53]. This strain is a BPSM-derivative gentamycin-resistant which allowsthe discrimination with BPZE1 (gentamycin-sensitive) on Bordet-Gengouagar plates containing 10 μg/ml of gentamycin and 100 μg/ml ofstreptomycin (Bogs). Control group corresponds to naive mice challengedwith BPSM/bctA-lacZ. One week after challenge infection, lungs wereaseptically removed, homogenized and plates on BGgs for CFUdetermination as previously described [18].

Mice were vaccinated with BPZE1 or aPv and challenged with virulent B.pertussis one, two, three or four weeks after vaccination. Lung CFUscounts were determined 3 hours or 7 days later. Results are expressed asmean (±standard error) CFUs from three to five mice per group. Levels ofprotection are calculated for each challenge infection as meanpercentages of CFUs of each group relative of the average of CFUs innon-immunized group, 7 days after challenge infection (Tables 2 to 5).

Example 10—Statistical Analysis

The results were analyzed using the unpaired Student's t test and theKruskal-Wallis test followed by the Dunn's post-test (GraphPad Prismprogram) when appropriate. Differences were considered significant atP≦0.05.

Results

Construction of B. pertussis BPZE1

Three virulence factors were genetically targeted: tracheal cytotoxin(TCT), pertussis toxin (PTX) and dermonecrotic toxin (DNT).

TCT is responsible for the destruction of ciliated cells in the tracheaof infected hosts [24, 25] and may thus be involved in the coughsyndrome. TCT is a breakdown product of peptidoglycan in the cell wallof Gram-negative bacteria, which generally internalize it into thecytosol by the AmpG transporter protein to be re-utilized during cellwall biosynthesis. B. pertussis AmpG is inefficient in theinternalization of peptidoglycan breakdown products. We thereforereplaced the B. pertussis ampG gene by E. coli ampG. The resultingstrain expressed less than 1% residual TCT activity (FIG. 1).

PTX is a major virulence factor responsible for the systemic effects ofB. pertussis infections and is composed of an enzymatically activemoiety, called S1, and a moiety responsible for binding to target cellreceptors (for review, see 26). However, it is also one of the majorprotective antigens, which has prompted us to replace the natural ptxgenes by a mutated version coding for an enzymatically inactive toxin.This was achieved by replacing Arg-9 by Lys and Glu-129 by Gly in S1,two key residues involved in substrate binding and catalysis,respectively. Allelic exchange was used to first delete the ptx operon,and then to insert the mutated version. The presence of the relevanttoxin analogues in the B. pertussis culture supernatants was evaluatedby immunoblot analysis (FIG. 2).

Finally, allelic exchange was used to remove the dnt gene (FIG. 3).Although the role of DNT in the virulence of B. pertussis is notcertain, it has been identified as an important toxin in the closelyrelated species Bordetella bronchiseptica and displays lethal activityupon injection of minute quantities (for review, see 26).

In Vitro Characterization of B. pertussis BPZE1

Since some of the genetic alterations in BPZE1 may potentially affectthe bacterial cell wall synthesis, the size and shape, as well as the invitro growth rate of BPZE1 was compared with those of the parentalstrain BPSM. The growth rate of BPZE1 did not differ from that of BPSM(FIG. 4), and no difference in bacterial shape or size was detectedbetween BPZE1 and BPSM, as evidenced by electron microscopy analysis(FIG. 5). However, the cell wall of BPZE1 appeared to be consistentlysomewhat thinner than that of BPSM.

To determine whether the absence or alterations of any of the targetedtoxins in BPZE1 affects adherence properties of B. pertussis, theattachment rates of BPZE1 was compared with those of BPSM, using thehuman pulmonary epithelial cell line A549 and the murine macrophage cellline J774, as two cellular models often used to study the adherence ofB. pertussis. No significant difference in the adherence capacities toeither cell line was observed between the two strains (FIG. 6).

Attenuation of B. pertussis BPZE

To determine whether the mutations introduced into B. pertussis BPZE1have resulted in attenuation, yet allow the organism to colonize therespiratory tract, Balb/C mice were intranasally infected with BPZE1 orBPSM, and colonization was followed over time. BPZE1 was able tocolonize and persist in the lungs of mice as long as BPSM (FIG. 7).However, the peak of multiplication seen 7 days after infection withBPSM was consistently lacking in mice infected with BPZE1. Studies donewith strains mutated in individual toxin genes indicated that this isdue to the mutations in the ptx locus (data not shown). When the lungswere examined for histopathological changes and inflammatoryinfiltration, infection with BPSM was found to induce strongperi-bronchiovascular infiltrates and inflammatory cell recruitment 7days after infection, associated with a strong hypertrophy of thebronchiolar epithelial cells (FIG. 8). In contrast, no such changes wereseen in BPZE1-infected animals, and the histology of the BPZE1-infectedmice was similar to that of the control mice that had received PBSinstead of the bacteria. The BPSM-infection induced inflammation lastedfor at least two months (data not shown). These results indicate thatthe mutations introduced into BPZE1 have resulted in drasticattenuation, but allow the bacteria to colonize and persist in thelungs.

Protection Against B. pertussis Challenge after Intranasal Vaccinationof Adult Mice with BPZE1

To evaluate the protection offered by BPZE1, the effect of a singleintranasal administration of this strain to 8-weeks old Balb/C mice onthe subsequent colonization by the wild type challenge strain BPSM wascompared with that of two i. p. immunizations with ⅕ of a human dose ofaPV. This aPV immunization protocol has been described as the best tocorrelate with pertussis vaccine efficacy in human clinical trials 127,281. As shown by the total clearance of bacterial colony counts in thelungs seven days after challenge infection, a single intranasaladministration of BPZE1 and two i.p. immunizations with aPV providedsimilar levels of protection (FIG. 9a ). High bacterial loads were foundin the control mice that had received two injections of PBS instead ofthe vaccine.

Protection Against B. pertussis Challenge after Intranasal Vaccinationof Infant Mice with BPZE1

Since the principal targets of novel pertussis vaccines are younginfants, that are not protected with the currently available vaccines,an infant (3 weeks-old) mouse model 1291 was developed and used tocompare the efficiency of vaccination with BPZE1 with that ofvaccination with aPV. A single nasal administration of BPZE1 fullyprotected infant mice against challenge infection (FIG. 9b ), ascomplete bacterial clearance was observed in the lungs one week afterchallenge. In contrast, substantial numbers of bacteria remained in theaPV-vaccinated animals one week after challenge infection. Thedifference in bacterial load between the BPZE1-vaccinated and theaPV-vaccinated mice was statistically significant, indicating that inthe infant mouse model a single intranasal administration with BPZE1provides better protection than two systemic administrations of aPV.

In addition, a strong reduction in the bacterial load of the challengestrain 3 hours after administration when the mice had been immunizedwith BPZE1 was consistently observed compared to the aPV-immunizedanimals (FIG. 9c ), indicating that vaccination with BPZE1 reduces thesusceptibility to infection by the challenge strain. This effect wasseen in both 8-weeks old and in infant mice. In contrast, aPV had noeffect on the bacterial counts 3 hours after infection, when compared tothe control mice.

Protection Against B. parapertussis Challenge after IntranasalVaccination with BPZE1

There is increasing concern about B. parapertussis infection inchildren, especially in immunized populations [30, 31]. B. parapertussiscauses a milder pertussis-like syndrome, the frequency of which isprobably largely underestimated. Furthermore, the incidence of B.parapertussis infections has been increasing over the last decades,possibly due to the fact that pertussis vaccines are known to have verylow or no protective efficacy against B. parapertussis (32, 331. Incontrast, infection by B. pertussis has recently been reported toprotect against B. parapertussis infection [34]. BPZE1 was also assessedfor protection against B. parapertussis using the infant mouse model.Whereas two administrations of aPV did not provide any protectionagainst B. parapertussis, as previously reported, a single intranasaladministration of BPZE1 provided strong protection, as measured by thelow numbers of B. parapertussis counts in the lungs of the vaccinatedmice 1 week after challenge (FIG. 9d ).

Immune Responses Induced by BPZE1 Vaccination

Although the mechanisms of protective immunity against B. pertussisinfection are not yet completely understood, clear evidence of a rolefor both B cells and IFN-γ has been demonstrated in mice [28].Vaccination with either one nasal dose of BPZE1 or two i. p.administrations of aPV induced high titers of serum IgG against FHA, amajor surface antigen of B. pertussis 1351, also present in aPV (FIG.10a ). Following B. pertussis challenge, positive anamnestic responseswere measured in BPZE1- and in aPV-vaccinated animals, as indicated byan increase in anti-FHA IgG titers, compared to primary responses beforeB. pertussis infection. Examination of the anti-FHA IgG1/IgG2a ratiosshowed that these ratios were higher after aPV administration,characteristic of a Th2 type response, than after BPZE1 vaccination(FIG. 10b ). Although the anti-FHA-IgG/IgG2a decreased after challengein the aPV vaccinated mice, it remained still substantially higher thanin the BPZE1-vaccinated animals after B. pertussis challenge.

Analysis of B. pertussis antigen-specific cytokine patterns induced byBPZE1 or aPV vaccination confirmed that BPZE1 administration favors astronger Th1 type response than aPV vaccination. This was revealed bythe fact that the ratios of IFN-γ over IL-5 produced by splenocytesstimulated with FHA or PT, or with the polyclonal activator ConA weresignificantly higher in BPZE1 vaccinated mice than in aPV vaccinatedmice (FIG. 10c ).

Protective Immunity of BPZE1 Over Time (from 1 Week to 4 Weeks)

As shown in Tables 1 to 5 below, whereas administration of aPv providedlimited protection (reduction of 75% of bacterial load compared tonon-vaccinated mice at 1 week) against B. pertussis, a single intranasaladministration of BPZE1 already provided high level of protection(reduction of 97.64% of bacterial load) against a B. pertussis challengeinfection performed one week after vaccination. If challenge infectionoccurred two weeks after vaccination, the level of protection induced byBPZE1 reached more than 99.999% compared to non-vaccinated mice and issignificantly superior to the protection induced by aPv vaccine(approximately 92% compared to non-vaccinated mice). Therefore, vaccineefficacy of BPZE1 against B. pertussis challenge is already significantone week after vaccination and is increasing over the at least next twomonths.

TABLE 1 Kinetics of vaccines efficacy against B. pertussis challenge ininfant mice. Time Time between between vac- lungs re- Log10 cfu/lungs ofmice cination and covery and aPv- BPZE1- challenge challenge Naivevaccinated vaccinated 1 week 3 hours 5.71 ± 0.03  5.8 ± 0.07 5.74 ± 0.017 days 6.71 ± 0.06 5.97 ± 0.20 4.86 ± 0.35 2 weeks 3 hours 5.77 ± 0.105.60 ± 0.02 5.49 ± 0.05 7 days 6.49 ± 0.08 5.31 ± 0.16 3.22 ± 0.33 3weeks 3 hours 6.03 ± 0.11 5.88 ± 0.04 5.33 ± 0.08 7 days 6.58 ± 0.095.62 ± 0.11 3.14 ± 0.38 4 weeks 3 hours 6.31 ± 0.01 6.15 ± 0.02 5.83 ±0.05 7 days 6.36 ± 0.04 5.21 ± 0.11 1.83 ± 0.46

TABLE 2 Level of protection of aPv-vaccinated and BPZE1-vaccinated miceas compared to non-vaccinated mice at week 1. Number of Mean number Nonvaccinated mice bacteria in lungs of bacteria Non-vaccinated 1 4.7 × 10⁶5.36 · 10⁶ Non-vaccinated 2 3.8 × 10⁶ Non-vaccinated 3 8.2 × 10⁶Non-vaccinated 4 4.1 × 10⁶ Non-vaccinated 5   6 × 10⁶ Mean Number ofPercentage percentage bacteria of remaining of remaining Level of inlungs bacteria ⁽¹⁾ bacteria protection aPv-vaccinated mice aPv1 1.95 ×10⁶  36.38   25%   75% aPv2 2.9 × 10⁶ 54.1 aPv3 2.9 × 10⁵ 5.41 aPv4 3.6× 10⁵ 6.72 aPv5 1.2 × 10⁶ 22.39 BPZE1-vaccinated mice BPZE1-1 3.2 × 10⁵5.97 2.36% 97.64% BPZE1-2   2 × 10⁴ 0.004 BPZE1-3   6 × 10⁴ 1.12 ⁽¹⁾Percentage of remaining bacteria = number of bacteria for eachparticular mouse/mean number of bacteria of all non-vaccinated mice

TABLE 3 Level of protection of aPv-vaccinated and BPZE1-vaccinated miceas compared to non-vaccinated mice at week 2. Number of Mean number Nonvaccinated mice bacteria in lungs of bacteria Non-vaccinated 1   5 × 10⁶3.34 × 10⁶ Non-vaccinated 2 3.6 × 10⁶ Non-vaccinated 3 1.7 × 10⁶Non-vaccinated 4 2.4 × 10⁶ Non-vaccinated 5   4 × 10⁶ Mean Number ofPercentage percentage of bacteria of remaining remaining Level of inlungs bacteria ⁽¹⁾ bacteria protection aPv-vaccinated mice aPv1 9.5 ×10⁴ 2.84 8.11%  91.89% aPv2 2.9 × 10⁵ 8.68 aPv3   1 × 10⁵ 2.99 aPv4 6.8× 10⁵ 20.36  aPv5 1.9 × 10⁵ 5.69 BPZE1-vaccinated mice BPZE1-1 9.5 × 10³ 2.8 × 10⁻³ 1.03 × 10⁻³% 99.999% BPZE1-2 450 1.35 × 10⁻⁴ BPZE1-3 3500 1.05 × 10⁻³ BPZE1-4 500  1.5 × 10⁻⁴ ⁽¹⁾ Percentage of remaining bacteria= number of bacteria for each particular mouse/mean number of bacteriaof all non-vaccinated mice

TABLE 4 Level of protection of aPv-vaccinated and BPZE1-vaccinated miceas compared to non-vaccinated mice at week 3. Number of Mean number Nonvaccinated mice bacteria in lungs of bacteria Non-vaccinated 1 1.8 × 10⁶4.04 × 10⁶ Non-vaccinated 2 5.75 × 10⁶  Non-vaccinated 3 4.7 × 10⁶Non-vaccinated 4 3.2 × 10⁶ Non-vaccinated 5 4.75 × 10⁶  Mean Number ofPercentage percentage of bacteria of remaining remaining Level of inlungs bacteria ⁽¹⁾ bacteria protection aPv-vaccinated mice aPv1 1.99 ×10⁵    4.94 11.26%  88.74% aPv2 6 × 10⁵ 14.85 aPv3 6 × 10⁵ 14.85 aPv44.2 × 10⁵   10.40 BPZE1-vaccinated mice BPZE1-1 3640 9.01 × 10⁻⁴ 8.65 ×10⁻⁴% 99.999% BPZE1-2 9720  2.4 × 10⁻³ BPZE1-3  300 7.43 × 10⁻⁵ BPZE1-4 340 8.42 × 10⁻⁵ ⁽¹⁾ Percentage of remaining bacteria = number ofbacteria for each particular mouse/mean number of bacteria of allnon-vaccinated mice

TABLE 5 Level of protection of aPv-vaccinated and BPZE1-vaccinated miceas compared to non-vaccinated mice at week 4. Number of Mean number Nonvaccinated mice bacteria in lungs of bacteria Non-vaccinated 1 2.1 × 10⁶2.36 × 10⁶ Non-vaccinated 2 2.2 × 10⁶ Non-vaccinated 3 3.1 × 10⁶Non-vaccinated 4 2.6 × 10⁶ Non-vaccinated 5 1.8 × 10⁶ Mean Number ofPercentage percentage of bacteria of remaining remaining Level of inlungs bacteria ⁽¹⁾ bacteria protection aPv-vaccinated mice aPv1 2.52 ×10⁵ 10.68  7.76%  92.24% aPv2 3.28 × 10⁵ 13.90  aPv3 1.04 × 10⁵ 4.41aPv4  8.4 × 10⁵ 3.56 aPv5 1.48 × 10⁵ 6.27 BPZE1-vaccinated mice BPZE1-1190 8.05 × 10⁻⁵ 7.13 × 10⁻⁵% 99.999% BPZE1-2  0 0   BPZE1-3 110 4.66 ×10⁻⁵ BPZE1-4 320 1.36 × 10⁻⁴ BPZE1-5 220 9.32 × 10⁻⁵ ⁽¹⁾ Percentage ofremaining bacteria = number of bacteria for each particular mouse/meannumber of bacteria of all non-vaccinated mice

Discussion

Pertussis is the first infectious disease whose incidence is increasingin countries with high vaccine coverage. This paradoxical situation ismost likely linked to the epidemiological changes observed since themassive introduction of highly efficacious vaccines. In contrast to thepre-vaccination era. cases of adolescent and adult pertussis are nowincreasingly more frequent. Although generally not life-threatening inthat age group, B. pertussis-infected adults are an important reservoirfor infection of the very young children, too young to be protected byvaccination. Early vaccination, possibly at birth, would therefore behighly desirable, but is hampered by the immaturity of the immune systemof neonates and infants. However, the fact that natural B. pertussisinfection, even very early in life, is able to induce a strong Th1response in infants [12] prompted us to develop a live attenuated B.pertussis vaccine strain to be given by the nasal route as analternative over the currently available vaccines.

Based on experimental infections of primates, Huang et al. had alreadyin 1962 come to the conclusion that ultimate protection against whoopingcough probably best follows a live B. pertussis inoculation [36]. Inveterinary medicine, attenuated Bordetella strains have been used tovaccinate against bordetellosis in dogs and piglets. A live attenuatedBordetella bronchiseptica strain has been shown to provide strongprotection against kennel cough in dogs 1371 after nasal administration.This protection was seen as early as 48 h after vaccination. Intranasalvaccination with live attenuated B. bronchiseptica has also been shownto protect against atrophic rhinitis in two-days old piglets [38],indicating that in a live attenuated form Bordetella vaccines can behighly active in new-born animals.

Previous attempts to genetically attenuate B. pertussis as a livevaccine candidate have met with rather limited success. Based on astrategy used for the successful attenuation of Salmonella vaccinestrains [39], Roberts et al. have deleted the aroA gene of B. pertussis[40]. The aroA mutant was indeed highly attenuated, but had also lostits capacity to colonize the respiratory tract of the intranasallyvaccinated animals and induced protective immunity only after repeatedadministrations of high doses. We took advantage of the knowledge on themolecular mechanisms of B. pertussis virulence and developed the highlyattenuated strain BPZE1. This strain contains genetic alterationsleading to the absence or inactivation of three major toxins, PTX, TCTand DNT. In contrast to the aroA mutant, this strain was able tocolonize the mouse respiratory tract and to provide full protectionafter a single intranasal administration. The protection in adult micewas indistinguishable from that induced by two administrations of ⅕ of ahuman dose of aPV. An important difference, however, was seen in infantmice, where a single administration of BPZE1 fully protected, whereasaPV only offered partial protection. In the context of the difficultiesto induce protection in infants with the currently available vaccinesearly in life, these results provide hope for the development of novelvaccination strategies that may be used in the very young children,possibly at birth. In addition, BPZE1 protected against B.parapertussis, whereas aPV did not. Therefore the use of BPZE1 shouldalso have an impact on the incidence of whooping cough caused by B.parapertussis in infants.

Although the recent replacement of first generation whole-cell vaccinesby new aPV in many countries has significantly reduced the systemicadverse reactions observed with whole-cell vaccines, it has notabolished the need for repeated vaccination to achieve protection. Thismakes it unlikely to obtain protection in very young children (<6months) that present the highest risk to develop severe disease. Inaddition, the wide-spread use of aPV has revealed new, unforeseenproblems. Repeated administration of aPV may cause extensive swelling atthe site of injection [41], which was not observed with whole-cellvaccines. In approximately 5% of the cases this swelling involves almostthe entire limb and lasts for more than a week. Although the mechanismof this swelling has not been characterized yet, it has been proposed tobe due to an Arthus hypersensitivity reaction caused by high antibodylevels induced by the primary immunization [42]. However, it could alsobe related to the Th2 skewing of the immune response, as, compared towhole-cell vaccines, aPV administration induces more Th2-type cytokinesin vaccinated children [10] and causes a delay in the Th1 development(Mascart et al., in preparation). Delayed maturation of Th1 function hasbeen associated with a risk for atopy in genetically pre-disposedindividuals [33]. The two mechanisms are not mutually exclusive.Compared to aPV, the immune response to BPZE1 administration is lessbiased towards the Th2 arm, and since BPZE1 is administered mucosally,no swelling reaction can occur.

The use of live attenuated bacteria as vaccines raises the issue oftheir biosafety. As such, they fall under the directives and guidelinesfor genetically modified organisms susceptible to be released into theenvironment. These guidelines and directives describe several objectivesthat have to be met, including hazard identification and environmentalrisk assessment [44]. Potential pathogenicity needs to be carefullyconsidered, especially in immuno-compromised individuals, such as thoseinfected with HIV. The natural biology of B. pertussis is particularlyinteresting in that regard. Although pertussis in HIV-infected subjectshas been described occasionally, it is rather rare in AIDS patients[45]. In its genetically attenuated form, B. pertussis would thereforenot be expected to cause major problems in HIV-infected children,especially if severe AIDS is an exclusion criterion, as it is for manyvaccines. B. pertussis colonization is strictly limited to therespiratory epithelium, without extrapulmonary dissemination of thebacteria, which naturally excludes systemic bacteremia of the BPZE1vaccine strain. If nevertheless unforeseeable safety problems occurred,the vaccine strain can easily be eliminated by the use of macrolideantibiotics. such as erythromycin, to which essentially all B. pertussisisolates are highly sensitive.

A further concern, like for any live vaccine, is the potential releaseof the vaccine strain in the environment and the consequences of such arelease. B. pertussis is a strictly human pathogen, and there is noanimal vector or reservoir. Moreover, unlike B. bronchiseptica, survivalof wild-type B. pertussis in the environment is extremely limited [46].Pertussis is only spread by coughing individuals, and there appears tobe no asymptomatic carriage [47]. Coughing cannot be assessed in themouse models used in this study. However, due to the nature of thegenetic alterations in BPZE1, in particular the strong reduction of TCTand the genetic inactivation of PTX, this strain would not be expectedto induce coughing. Active PTX has been shown to be required for coughinduction in a coughing rat model, although the mechanism is not known[48]. If the vaccine strain were nevertheless to be transmitted tonon-vaccinated individuals, this would at worst result in increasedvaccine coverage. The consequences of each of these potential hazardscan thus be graded as negligible and can easily and rapidly becontrolled by antibiotic treatment if necessary.

Advantages of the use of BPZE1 include the relatively low productioncosts, making it especially attractive for developing countries, itsneedle-free easy and safe mode of administration and its potential toinduce mucosal immunity in addition to systemic immunity. Although therole of mucosal immunity against pertussis has surprisingly not beenmuch addressed, the fact that B. pertussis is a strictly mucosalpathogen, makes it likely that mucosal immune responses may contributesignificantly to protection. None of the currently available vaccinesinduces any significant mucosal response.

Other advantages of the use of BPZE1 in vaccination are:

-   -   the rapid protective immune response obtained after a single        intranasal dose of BPZE1, since induction of the immunity can be        detected 1 week after vaccination,    -   an increase of the protective immunity over the at least next        two months after vaccination, and    -   the complete protective immunity, since a level of protection of        more than 99.999% is obtained 2 weeks after vaccination.

The use of live attenuated B. pertussis for mucosal vaccination offersyet another advantage. B. pertussis can be used for the presentation ofheterologous antigens to the respiratory mucosa (for review see 49). Theuse of BPZE1 as a heterologous expression platform may thus be helpfulfor the generation of multivalent vaccines against a variety ofrespiratory pathogens. However, since intranasal delivery of BPZE1 alsoinduces strong systemic immune responses, as shown here by both the highlevels of anti-FHA antibodies and of antigen-specific IFN-γ production,it may also be used for the production of antigens to which systemicimmune responses are desired.

While the invention has been described in terms of various preferredembodiments, the skilled artisan will appreciate that variousmodifications, substitutions, omissions and changes may be made withoutdeparting from the scope thereof. Accordingly, it is intended that thescope of the present invention be limited by the scope of the followingclaims, including equivalents thereof.

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What is claimed is:
 1. A vaccine comprising a pharmaceuticallyacceptable carrier and a dose of a live attenuated Bordetella strainwhich is able to colonize and induce protective immunity against amicrobial infection in a subject when administered to the subject. 2.The vaccine of claim 1, wherein the microbial infection is a Bordetellainfection.
 3. The vaccine of claim 1, wherein the live attenuatedBordetella pertussis strain comprises a heterologous expression platformfor carrying heterologous antigens.
 4. The vaccine of claim 3, whereinthe microbial infection is one caused by a respiratory pathogen selectedfrom the group consisting of: Neisseria, Pneumophila, Yersinia,Pseudomonas, Mycobacteria, and Influenza.
 5. The vaccine of claim 1,wherein the vaccine is formulated as a powder.
 6. The vaccine of claim1, wherein the vaccine is formulated as a liquid.
 7. The vaccine ofclaim 1, wherein the vaccine is formulated as a gel.
 8. A method ofmaking a vaccine comprising a pharmaceutically acceptable carrier and adose of a live attenuated Bordetella strain which is able to colonizeand induce protective immunity against a microbial infection in asubject when administered to the subject, the method comprising thesteps of: providing the live attenuated Bordetella strain, wherein thestrain comprises at least a mutated pertussis toxin (ptx) gene, adeleted or mutated dermonecrotic toxin (dnt) gene, and a heterologousampG gene replacing the Bordetella ampG gene; mixing the live attenuatedBordetella strain with a pharmaceutically acceptable carrier to yield avaccine mixture; and separating the vaccine mixture into single doses.